Human nucleotidases are enzymes that terminate purinergic receptor-mediated responses, including many processes dependent upon extracellular ATP and ADP. The potential clinical utility of injecting soluble nucleotidases therapeutically to modulate these physiologically important processes has already been demonstrated. One specific therapeutic use of such soluble nucleotidases is the control of the pathophysiological blood clotting that causes heart attacks and strokes, via hydrolysis of ADP that triggers platelet activation and subsequent blood coagulation. Thus, one goal of the proposed research is to elucidate the structures of the soluble human nucleotidases and to use this information to understand their functions. A second goal is to use the structural information to design modified nucleotidases, having increased activity and specificity for nucleotides modulating physiological processes. Elucidating the structural determinants of substrate hydrolysis and specificity is crucial for control of important, purinergically controlled, processes. The two known, naturally occurring, soluble forms of human ecto-nucleoside triphosphate diphosphohydrolases (eNTPDases) will be expressed in bacteria and refolded to generate large quantities of enzymatically active, soluble eNTPDases. The expressed proteins will be purified, enzymatically characterized, and crystallized to determine their structures. Affinity labeling, site-directed mutagenesis, and computer modeling will be used to determine the roles played by individual amino acid residues in nucleotide hydrolysis, as well as to identify residues important for nucleotidase specificity. These results will be compared and contrasted with similar results obtained using another soluble human nucleotidase, newly discovered by our laboratory, which is related in sequence to a nucleotidase found in the saliva of bloodsucking insects, but unrelated in sequence to the eNTPDases. The study of this nucleotidase is important for two reasons. First, it represents a second potential therapeutic avenue for developing therapeutic soluble nucleotidases. Second, it will demonstrate how two different protein primary structures are used to do the same enzymatic task, aiding in the design of more efficacious therapeutic soluble nucleotidases.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL072382-01A1
Application #
6687054
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Hasan, Ahmed AK
Project Start
2003-07-14
Project End
2007-05-31
Budget Start
2003-07-14
Budget End
2004-05-31
Support Year
1
Fiscal Year
2003
Total Cost
$357,875
Indirect Cost
Name
University of Cincinnati
Department
Pharmacology
Type
Schools of Medicine
DUNS #
041064767
City
Cincinnati
State
OH
Country
United States
Zip Code
45221
Herr, Andrew B; Conrady, Deborah G (2011) Thermodynamic analysis of metal ion-induced protein assembly. Methods Enzymol 488:101-21
Ivanenkov, Vasily V; Crawford, Patrick A; Toyama, Aimi et al. (2010) Epitope mapping in cell surface proteins by site-directed masking: defining the structural elements of NTPDase3 inhibition by a monoclonal antibody. Protein Eng Des Sel 23:579-88
Gaddie, Keith J; Kirley, Terence L (2009) Conserved polar residues stabilize transmembrane domains and promote oligomerization in human nucleoside triphosphate diphosphohydrolase 3. Biochemistry 48:9437-47
Ivanenkov, Vasily V; Sevigny, Jean; Kirley, Terence L (2008) Trafficking and intracellular ATPase activity of human ecto-nucleotidase NTPDase3 and the effect of ER-targeted NTPDase3 on protein folding. Biochemistry 47:9184-97
Yang, Mingyan; Kirley, Terence L (2008) Engineered human soluble calcium-activated nucleotidase inhibits coagulation in vitro and thrombosis in vivo. Thromb Res 122:541-8
Yang, Mingyan; Horii, Katsunori; Herr, Andrew B et al. (2008) Characterization and importance of the dimer interface of human calcium-activated nucleotidase. Biochemistry 47:771-8
Crawford, Patrick A; Gaddie, Keith J; Smith, Thomas M et al. (2007) Characterization of an alternative splice variant of human nucleoside triphosphate diphosphohydrolase 3 (NTPDase3): a possible modulator of nucleotidase activity and purinergic signaling. Arch Biochem Biophys 457:7-15
Kirley, Terence L; Crawford, Patrick A; Smith, Thomas M (2006) The structure of the nucleoside triphosphate diphosphohydrolases (NTPDases) as revealed by mutagenic and computational modeling analyses. Purinergic Signal 2:379-389
Murphy-Piedmonte, Deirdre M; Crawford, Patrick A; Kirley, Terence L (2005) Bacterial expression, folding, purification and characterization of soluble NTPDase5 (CD39L4) ecto-nucleotidase. Biochim Biophys Acta 1747:251-9
Ivanenkov, Vasily V; Murphy-Piedmonte, Deirdre M; Kirley, Terence L (2003) Bacterial expression, characterization, and disulfide bond determination of soluble human NTPDase6 (CD39L2) nucleotidase: implications for structure and function. Biochemistry 42:11726-35